Our long-term goal is to understand how cyclin-dependent kinase (CDK) inhibitors regulate cell division in the vertebrate. Cell division is one of the most fundamental processes in living organisms and understanding the regulation of cell cycle progression is critical for determining why cancer cells divide unchecked. In many cancer cells, negative cell cycle regulators such as CDK inhibitors are not expressed or are expressed at abnormally low levels. CDK inhibitors of the Cip/Kip-type are known to be regulated by protein turnover, but how their destruction is coordinated at the molecular level with the events of DNA replication initiation or environmental stress responses is still unclear. To understand the underlying mechanisms regulating CDK inhibitor function, we use eggs from the frog, Xenopus laevis, to study the relationship between CDK inhibitor proteolysis and DNA replication initiation. Our studies have indicated that the Xenopus Cip/Kip-type CDK inhibitor called p27 xicl (Xicl) is targeted for proteolysis only in the nucleus and in a manner dependent upon cell cycle phase, ubiquitination, and the proteasome. Our recent findings suggest that Xic1 proteolysis is regulated during the cell cycle by phosphorylation and during a DNA replication checkpoint. Mechanistically, we have demonstrated that Xic1 proteolysis is critically dependent upon binding to Proliferating Cell Nuclear Antigen (PCNA), a component of the DNA replication machinery. This suggests that Xic1 must be recruited to a site of initiation through its binding to PCNA before it is targeted for ubiquitination and degradation. Our findings thus link the proteolysis of Xic1 to DNA polymerase switching and the onset of processive DNA replication. On the basis of these findings, we hypothesize that Xic1 proteolysis is regulated during the cell cycle and during a cell cycle checkpoint by a PCNA- and DNA dependent mechanism at sites of replication initiation.
Our specific aims are: (1) To elucidate the molecular mechanism of Xic1 ubiquitination and degradation; (2) To define and characterize the regulation of Xic1 proteolysis during a response to DNA damage, a block to DNA replication, and the restart of DNA synthesis following DNA repair; and (3) To characterize how Xic1 phosphorylation and dephosphorylation regulates Xic1 proteolysis during the cell cycle. ? ?
